Peptide compositions and methods for treating patients

ABSTRACT

The present invention is directed to peptide compositions and methods of using the peptide compositions to treat prediabetes, diabetes, obesity, high blood pressure and metabolic syndrome.

This application is a divisional of U.S. patent application Ser. No.13/371,042 filed Feb. 10, 2012, which claims the benefit of the filingdate of U.S. Provisional Patent Application No. 61/441,748 filed Feb.11, 2011, which is incorporated by reference herein in its entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under HL68878 andHL89544 awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention is directed to peptide compositions and methods ofusing the peptide compositions to treat prediabetes, diabetes, obesity,high blood pressure and metabolic syndrome.

BACKGROUND

Our bodies turn the food we eat into the sugar glucose. Blood transportsglucose to cells which convert it into energy. Normally, a proteinhormone called insulin controls the level of glucose in the blood. Whenthere are defects in insulin production, insulin action, or both, highlevels of glucose in the blood result. Diabetes is the group of diseasescharacterized by these defects.

The three most common forms of diabetes are type 1 diabetes, type 2diabetes and gestational diabetes. Type 1 diabetes (previously known asinsulin-dependent diabetes mellitus or juvenile-onset diabetes) usuallydevelops in childhood or adolescence. It occurs when the body's immunesystem destroys the cells of the pancreas that produce insulin. Peoplewith type 1 diabetes must monitor the level of sugar in their bloodmultiple times a day and take insulin (via injections or a pump) tomaintain an appropriate level. Gestational diabetes occurs when pregnantwomen become intolerant to glucose. Gestational diabetes also requirestreatment to maintain appropriate glucose blood levels and avoidcomplications in the infant. Woman who have gestational diabetes are atincreased risk for developing type 2 diabetes.

Type 2 diabetes (previously known as non-insulin-dependent diabetesmellitus or adult-onset diabetes) usually develops in adulthood. Itdevelops as cells first do not use insulin properly and then thepancreas loses its ability to produce insulin. Many people with type 2diabetes control their blood glucose with a meal plan, exercise program,losing weight and taking oral medication. Some people with type 2diabetes need take insulin as well.

Diabetes is serious because too much sugar in the blood can damage theeyes, kidneys, nerves and heart. Complications of diabetes include heartdisease, stroke, hypertension, blindness, other eye problems (such asdiabetic retinopathy), kidney disease, nervous system disease (such asimpaired sensation or pain in the feet or hands, slowed digestion offood, carpal tunnel syndrome and erectile dysfunction), amputations,periodontal disease, susceptibility to other illnesses (such aspneumonia and influenza), impaired mobility and depression. Uncontrolleddiabetes can result in acute life-threatening events such as diabeticketoacidosis and hyperosmolar coma.

Diabetes is the leading cause of kidney failure, non-traumatic lowerlimb amputations and new cases of blindness among adults in the UnitedStates. Diabetes is a major cause of heart disease and stroke. Diabeteswas the seventh leading cause of death in the United States in 2007.Overall, the risk for death among people with diabetes is about twicethat of people of similar age without diabetes. According to the Centersfor Disease Control and Prevention, as of January 2011, diabetes affects25.8 million people, 8.3% of the United States population. Another 79million American adults are estimated to have prediabetes, a conditionin which blood sugar levels are higher than normal but not high enoughto be diagnosed as diabetes. Prediabetes is sometimes called impairedfasting glucose or impaired glucose tolerance. Prediabetes itself raisespeople's risk of type 2 diabetes, heart disease and stroke. Manyprediabetics develop type 2 diabetes within ten years.

In addition to lifestyle interventions, prediabetic and type 2 diabeticpatients are often treated with medications to address complications ofdiabetes. Doctors prescribe medications to control blood pressure andblood lipids to reduce cardiovascular complications. Often, in youngerand heavier patients with normal kidney function, doctors prescribe theoral drug metformin to more directly address the defects causingdiabetes. Metformin suppresses hepatic glucose production, increasesinsulin sensitivity, enhances peripheral glucose uptake, increases fattyacid oxidation and decreases absorption of glucose from thegastrointestinal tract. Metformin, though, is contraindicated in peoplewith any condition that could increase the risk of lactic acidosis,including kidney disorders, lung disease and liver disease.

Other more recently approved drugs do not appear to be more effectivethan metformin and each has its own set of contraindications. Forexample, rosiglitazone was one of the first insulin-sensitizers used asan anti-diabetic drug. It renders fat cells more sensitive to insulin.Annual sales of rosiglitazone peaked at approximately $2.5 billion in2006. Because rosiglitazone can be associated with an increased risk ofcardiovascular events, the European Medicines Agency recommended thedrug be suspended from the European market. The U.S. Food and DrugAgency has allowed it to remain on the market but it became subject tosignificant restrictions as of Sep. 23, 2010.

A precursor to insulin called human proinsulin C-peptide, and fragmentsof C-peptide, have also been investigated for the treatment of diabetes.See, International Publication Nos. WO 98/13384, WO 2002/022211, WO2004/016647, WO 2006/129095 and WO 2007/015069. See also, Ohtomo et al.,Diabetologia, 41: 287-291 (1998); Sato et al., Cell. Mol. Life. Sci.,61: 727-732 (2004); Hach et al., Exp. Diabetes Res.: 1-6 (2008) and Idoet al., Science, 277: 563-566 (1997).

Food proteins are composed of twenty different amino acids andscientists have studied the effect of individual amino acids wheningested with glucose. See, Gannon and Nuttall, IUBMB Life, 62: 660-668(2010); Gannon et al., Metabolism, 37: 1081-1088 (1988); Gannon et al.,Am. J. Clin. Nutr., 76: 1302-1307 (2002) and Kalogeropoulou et al.,Metabolism, 57: 1747-1752 (2008). The amino acids leucine and glycinehave been reported to attenuate the serum glucose response and stimulateadditional insulin secretion. This effect requires the ingestion ofsignificant amounts of the amino acids though, with accompanying badtaste, unbalanced amino acid intake and concerns of impairing renalfunction.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone. Incretinhormones are secreted by intestinal cells in response to nutrientingestion. The primary physiological function of GLP-1 appears to berelated to glycemic control. GLP-1 stimulates insulin release, inhibitsglucagon secretion, reduces gastric emptying and augments satiety. Inpatients with type 2 diabetes the incretin effect is reduced,contributing to impaired glycemic control. Administration of GLP-1 topatients has been reported to restore blood glucose regulation viaendogenous insulin secretion. GLP-1 administration has also beenreported to reduce energy intake through its actions of delaying gastricemptying and increasing satiety, therefore it may induce weight loss.Two GLP-receptor agonists/analogues are currently approved for treatmentof type 2 diabetes mellitus, exenatide (Byetta®), and liraglutide(Victoza®) and others are in clinical development. A once-weeklyformulation of exenatide (Bydureon®) has also been approved. See,Barnett et al., Diabetes, Obesity and Metabolism. accepted articlepublished online (2011).

In addition, studies have demonstrated that agonists of the GLP-1receptor also effect cardiovascular related functions such as heart rateand blood pressure. See, Grieve et al., British J. Pharm., 157a:1340-1351 (2009). In a particular study, Dahl salt-sensitive (DSS) ratswere fed a high salt diet and treated with an exenatide mimetic (AC3174)alone or in combination with an ACE inhibitor (captopril). AC3174 hadanti-hypertensive, insulin-sensitizing, and renoprotective effectscomparable to that of captopril. See, Liu et al., CardiovascularDiabetology, 9(32): 1-10 (2010).

There thus exists a need in the art for new treatments for prediabetes,diabetes and their complications. There also exists in the art a needfor new treatments for obesity, high blood pressure and metabolicsyndrome.

SUMMARY

The present invention provides products and methods for treatingprediabetes, diabetes, obesity, high blood pressure, metabolic syndrome,poor glycemic control, and reduced insulin secretion.

The invention provides a method for treating a condition comprisingadministering to a patient an effective amount of a compositioncomprising at least one peptide consisting of the amino acid sequenceGGL, GLG, LGL, LLG, LGG or GLL, or a pharmaceutically acceptable salt ofthe peptide, wherein the condition is prediabetes, diabetes, obesity,high blood pressure, metabolic syndrome, poor glycemic control, orreduced insulin secretion. In addition, the invention provides a methodfor treating a condition comprising administering to a patient aneffective amount of a composition comprising at least one peptideconsisting of the amino acid sequence GGdL, GdLG, GdLL, GLdL, GdLdL,dLLG, LdLG, dLdLG, dLGG, dLGL, LGdL, or dLGdL, or a pharmaceuticallyacceptable salt of the peptide, wherein the condition is prediabetes,diabetes, obesity, high blood pressure, metabolic syndrome, poorglycemic control, or reduced insulin secretion.

Also provided is a method of preventing, reducing, or ameliorating adiabetes-associated complication in a diabetic patient comprisingadministering to the patient an effective amount of a compositioncomprising at least one peptide consisting of the amino acid sequenceGGL, GLG, LGL, LLG, LGG or GLL, or a pharmaceutically acceptable salt ofthe peptide, wherein the diabetes-associated complication is acardiovascular disease, chronic kidney disease, kidney failure, bladderproblems, erectile dysfunction, gastroporesis, an eye disease, adiabetic neuropathy, foot or skin ulcers, or lower extremity amputation.In addition, the invention provides a method of preventing, reducing, orameliorating a diabetes-associated complication in a diabetic patientcomprising administering to the patient an effective amount of acomposition comprising at least one peptide consisting of the amino acidsequence GGdL, GdLG, GdLL, GLdL, GdLdL, dLLG, LdLG, dLdLG, dLGG, dLGL,LGdL or dLGdL, or a pharmaceutically acceptable salt of the peptide,wherein the diabetes-associated complication is a cardiovasculardisease, chronic kidney disease, kidney failure, bladder problems,erectile dysfunction, gastroporesis, an eye disease, a diabeticneuropathy, foot or skin ulcers, or lower extremity amputation.

In all of the foregoing methods, the peptides can be acetylated at theN-terminus, amidated at the C-terminus, or both. The composition can beadministered by an oral, intraperitoneal, ocular, intradermal,intranasal, subcutaneous, intramuscular or intravenous route.

The pharmaceutical compositions provided by the invention include acomposition comprising at least one peptide consisting of the amino acidsequence GGL, GLG, GLL, GGdL, GdLG, GdLL, GLdL, GdLdL, dLLG, LdLG,dLdLG, dLGG, dLGL, LGdL or dLGdL, or a pharmaceutically acceptable saltof the peptide, and a pharmaceutically acceptable excipient. They alsoinclude a composition wherein the pharmaceutical composition comprisesat least one peptide consisting of the amino acid sequence GGL, GLG, orGLL, or a pharmaceutically acceptable salt of the peptide, and apharmaceutically acceptable excipient. In the compositions, the peptidescan be acetylated at the N-terminus, amidated at the C-terminus, orboth.

The invention provides a kit for administering a pharmaceuticalcomposition comprising at least one peptide consisting of the amino acidsequence GGL, GLG, GLL, GGdL, GdLG, GdLL, GLdL, GdLdL, dLLG, LdLG,dLdLG, dLGG, dLGL, LGdL or dLGdL, or a pharmaceutically acceptable saltof the peptide, and a pharmaceutically acceptable excipient, wherein thekit comprises the composition, instructions for administration of thecomposition and a device for administering the composition to thepatient. Additionally, the invention provides a kit wherein thepharmaceutical composition comprises at least one peptide consisting ofthe amino acid sequence GGL, GLG, or GLL. In the kits, the peptides canbe acetylated at the N-terminus, amidated at the C-terminus, or both.

Cardiovascular diseases (CVD) are the primary cause of mortality amongdiabetic patients, accounting for almost two out of three deaths. Thus,minimization of risk of CVD is a critical clinical goal in themanagement of prediabetic and diabetic patients. The present inventionprovides products and methods that improve glycemic control andconcurrently decrease the risk of cardiovascular events and otherdiabetes-related complications.

BRIEF SUMMARY OF THE FIGURES

FIG. 1 shows the effect of glycine on blood glucose after oral loadglycine and glucose in C57BL/6J mice.

FIG. 2 shows the effect of leucine on blood glucose after oral loadleucine and glucose in C57BL/6J mice.

FIG. 3 shows Diapin inhibits the increase of blood glucose after oralload of glucose in KKay diabetic mice.

FIG. 4 show Diapin inhibits the increase of blood glucose after oralload of starch in KKay diabetic mice.

FIG. 5 shows Diapin reduces random blood glucose in KKay diabetic mice.

FIG. 6 shows Diapin stimulates insulin secretion in KKay diabetic mice30 min after oral load of glucose and Diapin.

FIG. 7 shows Diapin stimulates GLP-1 secretion in KKay diabetic mice 30min after oral load of glucose and Diapin.

FIG. 8 shows Diapin decreases the blood glucose level of KKay diabetesmouse in a time- and dose-dependent manner.

FIG. 9 shows Diapin has no effect on fasting blood glucose levels inC57BL/6J mice.

FIG. 10 shows Diapin inhibits the increase in blood glucose after the ipinjection of glucose into C57BL/6J mice.

FIG. 11 shows another peptide GGH has no effect on blood glucose afterthe ip injection of glucose into C57BL/6J mice.

FIG. 12 shows the effect of two other peptides, LGG and LGL, on bloodglucose after the ip injection of glucose into C57BL/6J mice.

FIG. 13 shows the effect of the peptide LLG on blood glucose after oralload of glucose in C57BL/6J mice.

FIG. 14 shows the effect of the peptides GLG and GLL on blood glucoseafter oral load of glucose in C57BL/6J mice.

FIG. 15 shows the effect of Diapin and Diapin with an amidatedC-terminus on blood glucose after oral administration of glucose inC57BL/6J mice.

FIG. 16 shows the effect of Diapin and Diapin with an acetylatedN-terminus on blood glucose after oral administration of glucose inC57BL/6J mice.

FIG. 17 shows the effect of Diapin with both an amidated C- andacetylated N-terminus on blood glucose after oral administration ofglucose in C57BL/6J mice.

FIG. 18 shows the effect of Diapin given at 30 min prior to oral glucoseadministration on blood glucose in C57BL/6J mice.

FIG. 19 shows the effect of Diapin given at 1 hour prior to oral glucoseadministration on blood glucose in C57BL/6J mice.

FIG. 20 shows the effect of Diapin and dipeptides on blood glucose levelin C57BL/6J mice after oral glucose administration.

FIG. 21 shows the effect of Diapin and dipeptides on blood glucose levelin C57BL/6J mice after oral glucose administration.

FIG. 22 shows the effect of Diapin in ob/ob mice after oral glucoseadministration.

FIG. 23 shows the effect of Diapin on blood glucose level in db/db miceafter oral glucose administration.

FIG. 24 shows the effect of Diapin on blood glucose level in high fatdiet-induced diabetic mice after oral glucose administration.

FIG. 25 shows the effect of D-Diapin (composed of D-isomer amino acids)on lowering blood glucose level in C57BL/6J mice after oral glucoseadministration.

FIG. 26 shows the effect of Diapin on blood glucose level in C57BL/6Jmice after oral glucose administration and ip Diapin administration.

FIG. 27 shows the effect of modified dipeptides on blood glucose levelin C57BL/6J mice after oral glucose administration.

DETAILED DESCRIPTION

In one aspect, the invention provides peptides to be administered toprediabetic or diabetic patients. Examples of peptides of the inventionare GGL (termed “Diapin” herein), GLG, LGL, LLG, LGG and GLL. Otherexamples of peptides of the invention are GL and LG. The invention alsoprovides for peptides GGL, GLG, LGL, LLG, LGG and GLL in which eachleucine is independently in the form of the L-isomer or the D-isomer.Other examples of the peptides of the invention are LG and GL in whichleucine is in the D-isomeric form. Peptides of the invention may bechemically synthesized or derived by digestion of proteins by methodsknown in the art.

As used herein, the singular forms “a”, “an”, and “the” include pluralreferences unless the context clearly dictates otherwise.

It is known in the art that it is possible to substitute a chemicallysimilar amino acid for an amino acid in a peptide or protein withoutnegatively affecting the activity of the peptide or protein. Therefore,it is specifically contemplated that a glycine or leucine residue in apeptide of the invention may be substituted with a chemically similaramino acid residue such as a different aliphatic amino acid residue oran amino acid isomer. Other aliphatic amino acids are alanine, valineand isoleucine. It is also specifically contemplated that chemicallysimilar amino acids may be added to one or both ends of a peptide of theinvention without negatively affecting the activity of the peptide.

With the exception of glycine, the common amino acids all contain atleast one chiral carbon atom. These amino acids therefore exist as pairsof stereoisomers designated as the L-isomer and the D-isomer. Mostnaturally occurring proteins and peptides are composed exclusively ofthe L-isomeric form. D-isomeric amino acids can affect the conformationof a peptide or protein and may lead to increased stability or a changein activity.

In some embodiments of the peptide, Leucine is replaced with D-Leucine.For example in some embodiments the peptide is Glycine-Glycine-Leucine(GGL), or is Glycine-Glycine-D-Leucine (GGdL), or isGlycine-Leucine-Glycine (GLG), or is Glycine-D-Leucine-Glycine (GdLG),or is Leucine-Leucine-Glycine (LLG), or is D-Leucine-Leucine-Glycine(dLLG), or is Leucine-D-Leucine-Glycine (LdLG), or isD-Leucine-D-Leucine-Glycine (dLdLG), or is Leucine-Leucine-Glycine(LLG), or is D-Leucine-Leucine-Glycine (dLLG), or isLeucine-D-Leucine-Glycine (LdLG), or is D-Leucine-D-Leucine-Glycine(dLdLG), or is Leucine-Glycine-Glycine (LGG), or isD-Leucine-Glycine-Glycine (dLGG), or is Glycine-Leucine-Leucine (GLL),or is Glycine-D-Leucine-Leucine (GdLL), or is Glycine-Leucine-D-Leucine(GLdL), or is Glycine-D-Leucine-D-Leucine (GdLdL), or is Leucine-Glycine(LG), or is D-Leucine-Glycine (dLG), or is Glycine-Leucine (GL), or isGlycine-D-Leucine (GdL). The peptides of the invention may be usedindividually or used as a mixture of two or more peptides. With respectto a mixture, each possible subcombination of peptides is specificallycontemplated by the invention.

In some embodiments, peptides of the invention are chemically modified.In some embodiments peptides of the invention are acetylated at theN-terminus. In some embodiments, peptides of the invention are amidatedat the C-terminus. In some embodiments, peptides of the invention areacetylated at the N-terminus and amidated at the C-terminus. Peptidesare acetylated or amidated by methods known in the art. In someembodiments of the present disclosure, the peptide is glycosylated,carboxylated, phosphorylated, esterified, or converted into an acidaddition salt and/or optionally dimerized, polymerized, pegylated, orotherwise conjugated.

In some embodiments, the peptides comprise one or more non-peptide bondsin place of peptide bond(s). For example, the peptides comprise in placeof a peptide bond, an ester bond, an ether bond, a thioether bond or anamide bond.

In another aspect, compositions of at least one of the peptides of theinvention are provided. Examples of compositions of the invention arecompositions comprising one or more of the peptides GGL, GLG, LGL, LLG,LGG and GLL, or a pharmaceutically acceptable salt thereof. Otherexamples of compositions of the invention are compositions comprisingone or more of the peptides GL and LG, or pharmaceutically acceptablesalts thereof. Other examples of compositions of the invention arecompositions comprising GGL, GLG, GLL, LLG, LGG, LGL, GGdL, GdLG, GdLL,GLdL, GdLdL, dLLG, LdLG, dLdLG, dLGG, dLGL, LGdL, dLGdL, or apharmaceutically acceptable salt thereof. The compositions of theinvention may include other components, including other amino acids.With respect to the compositions, each possible subcombination ofpeptides is specifically contemplated by the invention.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. A“pharmaceutically acceptable salt” means any non-toxic salt or salt ofan ester of a compound of this invention that, upon administration to arecipient, is capable of providing, either directly or indirectly, acompound of this invention. Pharmaceutically acceptable salts are wellknown in the art. For example, Berge et al. describe pharmaceuticallyacceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19(1977).

It is contemplated the peptides of the invention, or mixtures thereof,can be used as the sole active product ingredient in the composition.Accordingly in an aspect of the invention, compositions of one or moreof the peptides of the invention are provided wherein the peptide orpeptides of the invention are the sole active ingredient. Thus, anembodiment of the present disclosure is a composition consistingessentially of at least one peptide consisting of the amino acidsequence GGL, GLG, LGL, LLG, LGG or GLL, or pharmaceutically acceptablesalts thereof. Another embodiment is a composition consistingessentially of at least one peptide consisting of the amino acidsequence GL or LG, or pharmaceutically acceptable salts thereof. Yetanother embodiment is a composition consisting essentially of at leastone peptide consisting of the amino acid sequence GGL, GLG, GLL, LLG,LGG, LGL, GGdL, GdLG, GdLL, GLdL, GdLdL, dLLG, LdLG, dLdLG, dLGG, dLGL,LGdL, dLGdL, or a pharmaceutically acceptable salt thereof. With respectto the compositions, each possible subcombination of peptides isspecifically contemplated by the invention.

In yet a further aspect, the invention provides a composition comprisingat least one peptide of the invention and a pharmaceutically acceptableexcipient.

In some embodiments, a pharmaceutical composition comprises at least onepeptide consisting of the amino acid sequence GGL, GLG, GLL, GGdL, GdLG,GdLL, GLdL, GdLdL, dLLG, LdLG, dLdLG, dLGG, dLGL, LGdL or dLGdL, or apharmaceutically acceptable salt of the peptide, and a pharmaceuticallyacceptable excipient. In some embodiments, the pharmaceuticalcomposition comprises at least one peptide consisting of the amino acidsequence GGL, GLG, or GLL, or a pharmaceutically acceptable salt of thepeptide, and a pharmaceutically acceptable excipient. In someembodiments, the pharmaceutical composition comprises the peptideconsisting of the amino acid sequence GGL, or a pharmaceuticallyacceptable salt of the peptide, and a pharmaceutically acceptableexcipient. With respect to the pharmaceutical compositions, eachpossible subcombination of peptides is specifically contemplated by theinvention.

Pharmaceutical compositions of the invention are formulated withpharmaceutically acceptable excipients such as carriers, solvents,stabilizers, adjuvants, diluents, etc., depending upon the particularmode of administration and dosage form. The compositions are generallyformulated to achieve a physiologically compatible pH, and range from apH of about 3 to a pH of about 11, about pH 3 to about pH 7, dependingon the formulation and route of administration. In alternativeembodiments, the pH is adjusted to a range from about pH 5.0 to about pH8. In various aspects, the compositions comprise a therapeuticallyeffective amount of at least one peptide as described herein, togetherwith one or more pharmaceutically acceptable excipients. Thecompositions may include a second active ingredient useful in thetreatment or prevention of bacterial growth (for example and withoutlimitation, anti-bacterial or anti-microbial agents).

Suitable excipients include, for example, carrier molecules that includelarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, and inactive virus particles. Otherexemplary excipients include antioxidants (for example and withoutlimitation, ascorbic acid), chelating agents (for example and withoutlimitation, EDTA), carbohydrates (for example and without limitation,dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose),stearic acid, liquids (for example and without limitation, oils, water,saline, glycerol and ethanol) wetting or emulsifying agents, pHbuffering substances, and the like.

Pharmaceutical compositions suitable for the delivery of peptides of thepresent invention and methods for their preparation will be readilyapparent to those skilled in the art. Such compositions and methods fortheir preparation may be found, for example, in Remington'sPharmaceutical Sciences, The Science and Practice of Pharmacy, 20thEdition, Lippincott Williams & White, Baltimore, Md. (2000). Thepeptides of the present invention may be formulated to be immediateand/or modified release.

In yet another aspect, the invention provides a method for treating acondition comprising administering to a patient an effective amount of acomposition comprising at least one peptide consisting of the amino acidsequence GGL, GLG, LGL, LLG, LGG or GLL, or a pharmaceuticallyacceptable salt of the peptide, wherein the condition is prediabetes,diabetes, obesity, high blood pressure, metabolic syndrome, poorglycemic control, or reduced insulin secretion. The invention alsoprovides a method for treating a condition comprising administering to apatient an effective amount of a composition comprising at least onepeptide consisting of the amino acid sequence GGdL, GdLG, GdLL, GLdL,GdLdL, dLLG, LdLG, dLdLG, dLGG, dLGL, LGdL, or dLGdL, or apharmaceutically acceptable salt of the peptide, wherein the conditionis prediabetes, diabetes, obesity, high blood pressure, metabolicsyndrome, poor glycemic control, or reduced insulin secretion. Withrespect to the methods, each possible subcombination of peptides isspecifically contemplated by the invention.

In some embodiments, the invention provides a method for treating aprediabetic or diabetic patient comprises administering to the patient acomposition comprising at least one of the peptides GGL, GLG, LGL, LLGand GLL. In some embodiments, the invention also provides a method fortreating a prediabetic or diabetic patient comprising administering tothe patient a composition comprising at least one of peptide GL or LG.In some embodiments, the diabetes is type 1 diabetes. In someembodiments, the diabetes is type 2 diabetes. The amount of thecomposition administered is therapeutically effective to achieve atleast one of the following: reducing blood glucose levels, stimulatinginsulin secretion, stimulating GLP-1 secretion, reducing insulinresistance, and improving glycemic control.

The term “treating” (or other forms of the word such as “treatment” or“treat”) is used herein to mean that administration of a composition ofthe present invention mitigates a condition in a patient and/or reduces,inhibits, or eliminates a particular characteristic or event associatedwith a condition. Thus, the term “treatment” includes, preventing acondition from occurring in a patient, particularly when the patient ispredisposed to acquiring the condition; reducing or inhibiting thecondition; and/or ameliorating or reversing the condition. Insofar asthe methods of the present invention are directed to preventingconditions, it is understood that the term “prevent” does not requirethat the condition be completely thwarted. Rather, as used herein, theterm preventing refers to the ability of the skilled artisan to identifya population that is susceptible to condition, such that administrationof the compositions of the present invention may occur prior to onset ofthe condition. The term does not imply that the condition must becompletely avoided.

An “effective amount” as used herein refers to an amount of a peptide ofthe invention sufficient to exhibit a detectable therapeutic effect. Theeffect is detected by, for example, an improvement in clinicalcondition, or a prevention, reduction or amelioration of complications.The precise effective amount for a patient will depend upon thepatient's body weight, size, and health; the nature and extent of thecondition; and the therapeutic or combination of therapeutics selectedfor administration. Therapeutically effective amounts for a givensituation are determined by routine experimentation that is within theskill and judgment of the clinician.

In some embodiments, the invention provides methods for treatingobesity, high blood pressure or metabolic syndrome. Accordingly, oneembodiment of the invention is a method for treating obesity comprisingadministering to a patient an effective amount of composition comprisingat least one peptide consisting of the amino acid sequence GGL, GLG,LGL, LLG, LGG, GLL, LG, or GL, or a pharmaceutically acceptable saltthereof. Still another embodiment is a method for treating high bloodpressure comprising administering to a patient an effective amount of acomposition comprising at least one peptide consisting of the amino acidsequence GGL, GLG, LGL, LLG, LGG, GLL, LG, or GL, or a pharmaceuticallyacceptable salt thereof. Another embodiment is a method for treatingmetabolic syndrome comprising administering to a patient an effectiveamount of a composition comprising at least one peptide consisting ofthe amino acid sequence GGL, GLG, LGL, LLG, LGG, GLL, LG, or GL, or apharmaceutically acceptable salt thereof. In any of the foregoingembodiments, one or more leucine in the peptide is independentlyreplaced with the D-isomer of leucine. With respect to the methods, eachpossible subcombination of peptides is specifically contemplated by theinvention.

In still another aspect, the invention provides methods for preventing,reducing and/or ameliorating diabetes-associated complications in aprediabetic or diabetic patient comprising administering to the patienta composition comprising at least one peptide consisting of the aminoacid sequence GGL, GLG, LGL, LLG, LGG or GLL. The invention alsoprovides methods for preventing, reducing and/or amelioratingdiabetes-associated complications in a prediabetic or diabetic patientcomprising administering to the patient a composition comprising atleast one of the peptides consisting of the amino acid sequence GL orLG. It also provides a method of preventing, reducing, or ameliorating adiabetes-associated complication in a diabetic patient comprisingadministering to the patient an effective amount of a compositioncomprising at least one peptide consisting of the amino acid sequenceGGL, GLG, LGL, LLG, LGG or GLL, or a pharmaceutically acceptable salt ofthe peptide. It also provides a method of preventing, reducing, orameliorating a diabetes-associated complication in a diabetic patientcomprising administering to the patient an effective amount of acomposition comprising at least one peptide consisting of the amino acidsequence GGdL, GdLG, GdLL, GLdL, GdLdL, dLLG, LdLG, dLdLG, dLGG, dLGL,LGdL or dLGdL, or a pharmaceutically acceptable salt of the peptide.With respect to the methods, each possible subcombination of peptides isspecifically contemplated by the invention. In some embodiments, thediabetes is type 1 diabetes. In some embodiments, the diabetes is type 2diabetes. The administration is of an amount of the composition that istherapeutically effective to prevent, reduce or ameliorate at least onediabetes-associated complication including, but not limited to, thefollowing: a cardiovascular disease [e.g., coronary artery disease(sometimes called ischemic heart disease), cerebral vascular diseases(such as stroke or transient ischemic attacks), heart failure,atherosclerosis, or peripheral arterial disease], chronic kidneydisease, kidney failure, bladder problems, erectile dysfunction,gastroporesis, an eye disease (such as diabetic retinopathy, cataract orglaucoma), a diabetic neuropathy (peripheral, autonomic, proximal orfocal), foot or skin ulcers, or lower extremity amputation.

The compounds of the present invention may be administered by anysuitable route. For example, compositions of the invention can beadministered by the oral, ocular, intradermal, intraperitoneal (“ip”),intranasal, subcutaneous, intramuscular or intravenous route.

Formulations suitable for oral administration include, for example,solid, semi-solid and liquid systems such as, tablets; soft or hardcapsules containing multi- or nano-particulates, liquids, or powders;lozenges (including liquid-filled); chews; gels; fast dispersing dosageforms; films; ovules; sprays. In some embodiments the peptides of thepresent invention are formulated for oral administration using deliveryvehicles known in the art, including but not limited to, microspheres,liposomes, enteric coated dry emulsions or nanoparticles.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active peptides, theliquid dosage forms may contain inert diluents commonly used in the artsuch as, for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activepeptide is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents. The active compounds canalso be in microencapsulated form with one or more excipients as notedabove. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugar as well as high molecular weight polyethyleneglycols and the like. The solid dosage forms of tablets, dragees,capsules, pills, and granules can be prepared with coatings and shellssuch as enteric coatings and other coatings well known in thepharmaceutical formulating art. Injectable preparations, for example,sterile injectable aqueous or oleaginous suspensions may be formulatedaccording to the known art using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation may also be asterile injectable solution, suspension or emulsion in a nontoxicparenterally acceptable diluent or solvent, for example, as a solutionin 1,3-butanediol. Among the acceptable vehicles and solvents that maybe employed are water, Ringer's solution, U.S.P. and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose any blandfixed oil can be employed including synthetic mono- or diglycerides. Inaddition, fatty acids such as oleic acid are used in the preparation ofinjectables. The injectable formulations can be sterilized, for example,by filtration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Treatment of pre-diabetic or diabetic patients with compositions of theinvention in combination with other diabetes drugs known in the art isspecifically contemplated. In some embodiments, treatment withcompositions of the invention allows a reduction in the dose of theother diabetes drug or drugs and therefore reduces the side effectsassociated with the other drug or drugs. In some embodiments, the otherdiabetes drug is insulin. In some embodiments, the other diabetes drugis a biguanide (such as metformin). In some embodiments, the otherdiabetes drug is a thiazolidinedione (such as pioglitazone). In someembodiments, the other diabetes drug is a DPP-4 inhibitor (such assitagliptin). In other words, compositions of the present invention canbe used in combination with other drugs such as those used as standardof care for the condition being treated. In some embodiments the drug isa statin (including but not limited to, atorvastatin, lovastatin,simvastatin, pravastatin rosuvastatin, fluvastatin, and pitastatin). Insome embodiments, the drug is a blood pressure lowering drug [includingbut not limited to, Angiotensin-converting enzyme (ACE) inhibitors suchas captopril, lisinopril, and ramipril; Angiotensin II receptor blockerssuch as losartan, olmesartan and valsartan; beta blockers such asmetoprolol, nadolol and penbutolol; and calcium channel blockers such asamlodipine, diltiazem and nifedipine].

In still another aspect, the invention provides a kit for administeringa composition of invention to a patient in need thereof, where the kitcomprises a composition of invention, instructions for use of thecomposition and a device for administering the composition to thepatient. In some embodiments, a kit for administering a pharmaceuticalcomposition comprises at least one peptide consisting of the amino acidsequence GGL, GLG, GLL, LLG, LGL, LGG, GGdL, GdLG, GdLL, GLdL, GdLdL,dLLG, LdLG, dLdLG, dLGG, dLGL, LGdL or dLGdL, or a pharmaceuticallyacceptable salt of the peptide, and a pharmaceutically acceptableexcipient, wherein the kit comprises the composition, instructions foradministration of the composition and a device for administering thecomposition to the patient. In some embodiments, the kit comprises apharmaceutical composition comprising at least one peptide consisting ofthe amino acid sequence GGL, GLG, or GLL. With respect to the kits, eachpossible subcombination of peptides is specifically contemplated by theinvention.

EXAMPLES

The invention will be more fully understood by reference to thefollowing examples which detail exemplary embodiments of the invention.

Example 1

The effects of the amino acids glycine and leucine on blood glucose weredetermined.

Effect of Glycine on Blood Glucose after Oral Load Glycine and Glucose

An experiment was performed in adult male C57BL/6J mice purchased fromJackson Lab (Bar Harbor, Me.). Fasted mice were given glucose (2 mg/gbody weight, diamond line in FIG. 1, n=10) or glycine (0.35 mg/g bw,square line in FIG. 1, n=10) and glucose by gavaging. Blood glucose wasmeasured at 0, 30, 60, 90, and 120 min after giving glucose.

Blood glucose levels at any time point in the glycine group were notsignificantly changed compared to the control group.

Effect of Leucine on Blood Glucose after Oral Load Leucine and Glucose

An experiment was performed in adult male C57BL/6J mice purchased fromJackson Lab. Fasted mice were given glucose (2 mg/g body weight, diamondline in FIG. 2, n=10) or Leucine (0.35 mg/g bw, square line in FIG. 2,n=10) and glucose by gavaging. Blood glucose was measured at 0, 30, 60,90, 120 min after giving glucose.

Example 2

Diapin (peptide GGL of the invention) potently attenuates blood glucoselevels when orally ingested with either glucose or starch in a diabeticmouse. Moreover, Diapin also reduces blood glucose levels undernon-fasting condition in KKay diabetic mice [Yamauchi et al., Nat. Med.,7(8): 971-946 (2001)]. See FIG. 3.

Diapin Inhibits the Increase of Blood Glucose after Oral Load of Glucosein Diabetic Mice

Blood glucose levels at 30, 60, 90 and 120 min in Diapin group wassignificantly lower than those in the control.

Diapin Inhibits the Increase in Blood Glucose after Oral Load of Starchin Diabetic Mice

An experiment was performed in adult male KKay diabetic mice purchasedfrom the Jackson Lab. In the control group (diamond line in FIG. 4,n=10), starch was orally administered at dose of 2 mg/g bw. In theDiapin group (square line in FIG. 4, n=9), starch and Diapin were orallyadministered at dose of 2 mg/g bw and 1 mg/g bw, respectively. Bloodglucose was measured at 0, 30, 60, 90 and 120 min after gavaging starchand Diapin.

Blood glucose levels at 30, 60, 90 and 120 min in the Diapin group wassignificantly lower than those in the control.

Diapin Reduces Random Blood Glucose in Diabetic Mice

An experiment was performed in adult male KKay diabetic mice purchasedfrom the Jackson Lab. Under non-fasting condition, in the control group(diamond line in FIG. 5, n=9), distilled water was orally given and inthe Diapin group (square line in FIG. 5, n=9), Diapin was orallyadministered at 1 mg/g bw. Blood glucose was measured at 0, 30, 60, 90,120, 150 and 180 min after gavaging of Diapin.

Blood glucose levels at 60, 90, 120, 150 and 180 min in the Diapin groupwere significantly lower than those in the control.

Example 3

Diapin per se stimulates insulin secretion in KKay diabetic mice.Furthermore, Diapin also increases GLP-1 secretion in diabetic mice.

Diapin Stimulates Insulin Secretion in Diabetic Mice after Oral Load ofGlucose and Diapin

An experiment was performed in adult male KKay diabetic mice. Underfasting conditions in the control group (white bar in FIG. 6, n=11),glucose was orally administered at dose of 1.5 mg/g bw. In the Diapingroup (black bar in FIG. 6, n=11), Diapin and glucose were orallyadministered at 1 mg/g bw and 1.5 mg/g bw, respectively. Blood sampleswere collected at 30 min after oral administration of glucose andDiapin. Blood glucose was monitored with FreeStyle glucose meter andinsulin was measured by ELISA (Alpco, Cat#80-INSMS-E01).

Diapin stimulated insulin secretion in the KKay diabetic mice.

Diapin Stimulates GLP-1 Secretion in Diabetic Mice after Oral Load ofGlucose and Diapin

An experiment was performed in adult male KKay diabetic mice. Underfasting conditions, in the control group (white bar in FIG. 7, n=11),glucose was orally administered at dose of 1.5 mg/g bw and in the Diapingroup (black bar in FIG. 7, n=11), Diapin and glucose were orallyadministered at 1 mg/g bw and 1.5 mg/g bw, respectively. Blood sampleswere collected at 30 min after oral administration of glucose andDiapin. GLP-1 was measured by ELISA (Alpco, Cat#43-GP1HU-E01).

Diapin also increases GLP-1 secretion in diabetic mice.

Example 4

Diapin ingested with diet decreases random blood glucose levels in KKaydiabetic mice in a time- and dose-dependent manner.

An experiment was performed in adult male KKay diabetic mice. The micewere divided into three groups of 10 animals each and fed, ad libitum,regular chow (control), chow, chow mixed with 6 g Diapin/kg, or chowmixed with 12 g Diapin/kg for the duration of the experiment. Bloodglucose levels were measured weekly in the early morning at initiation(week 0), and weekly thereafter for 4 weeks. Results are shown in FIG.8.

Blood glucose levels in both groups fed with chow mixed with Diapin weresignificantly lower than those in the control.

Example 5

Diapin does not reduce blood glucose levels in non-diabetic C57BL/6Jmice when blood glucose levels are at normal levels.

Diapin has No Effect on Fasting Blood Glucose Levels in C57BL/6J Mice

An experiment was performed in adult male C57BL/6J mice purchased fromJackson Lab. The mice were given water (diamond line in FIG. 9, n=6) orDiapin (2 mg/g bw, square line in FIG. 9, n=6). Blood glucose wasmeasured at 0, 30, 60, 90, 120, 150 and 180 min after oraladministration of Diapin.

There was no significant difference in blood glucose levels between thegroups.

Example 6

Diapin, LGL, LGG reduce blood glucose levels in non-diabetic C57BL/6Jmice after glucose is loaded intraperitoneally. In comparison, thepeptide GGH does not.

Diapin Inhibits the Increase of Blood Glucose after the Ip Injection ofGlucose

An experiment was performed in adult male C57BL/6J mice purchased fromJackson Lab. Fasted mice were given water (diamond line in FIG. 10,n=10) or Diapin orally (1 mg/g bw, square line in FIG. 10, n=10).Glucose was given by ip injection at 10 minutes after the oraladministration of Diapin. Blood glucose was measured at 0, 30, 60, 90,120 min after giving glucose.

Blood glucose levels at 30, 60, 90 and 120 min in Diapin group weresignificantly lower than those in the control.

The Peptide GGH has No Significant Effect on Blood Glucose after the IpInjection of Glucose

An experiment was performed in adult male C57BL/6J mice purchased fromJackson Lab. Fasted mice were given water (diamond line in FIG. 11,n=10) or GGH (1 mg/g bw, square line in FIG. 11, n=10). Glucose wasgiven by ip injection at 10 minutes after oral administration of GGH.Blood glucose was measured at 0, 30, 60, 90, 120 min after givingglucose.

Blood glucose levels at any time point in the GGH group were notsignificantly changed compared to the control mice.

The Peptides LGG and LGL Inhibit the Increase in Blood Glucose after theIp Injection of Glucose

An experiment was performed in adult male C57BL/6J mice purchased fromJackson Lab. Fasted mice were given water (diamond line in FIG. 12,n=10) or LGL (1 mg/g bw, square line in FIG. 12, n=10) or LGG (1 mg/gbw, triange line in FIG. 12, n=10). Glucose was given by ip injection 10minutes after the oral administration of LGG or LGL. Blood glucose wasmeasured at 0, 30, 60, 90, 120 min after giving glucose.

Blood glucose levels at 30, 60, 90 and 120 min in LGL group aresignificantly lower than those in the control. The peptide LGG reducesthe blood glucose levels at 30 and 60 min.

Example 7

The peptides LGL, GLG, LLG, and GLL significantly reduce blood glucoselevels in non-diabetic C57BL/6J mice loaded with glucose.

Effect of LLG on Blood Glucose after Oral Load of Glucose

An experiment was performed in adult male C57BL/6J mice purchased fromJackson Lab. In the control group (diamond line in FIG. 13, n=10),glucose was orally administered at dose of 2 mg/g bw. In the Diapin andLLG group (square line or triangle line, n=9), glucose and Diapin orglucose and LLG were orally administered at dose of 2 mg/g bw and 1 mg/gbw, respectively. Blood glucose was measured at 0, 30, 60, 90 and 120min after gavaging glucose and Diapin.

LLG showed similar effects to Diapin.

Effect of Peptides GLG and GLL on Blood Glucose after Oral Load ofGlucose

An experiment was performed in adult male C57BL/6J mice purchased fromJackson Lab. In the control group (diamond line in FIG. 14, n=10),glucose was orally administered at dose of 2 mg/g bw. In the GLG and GLLgroup (triangle line or circle line in FIG. 14, n=9), glucose and GLG orGLL were orally administered at dose of 2 mg/g bw and 1 mg/g bw,respectively. Blood glucose was measured at 0, 30, 60, 90 and 120 minafter gavaging glucose and GLG or GLL.

Peptides GLG and GLL each showed similar effects to Diapin.

Example 8

Amidation and acetylation do not decrease Diapin glucose reduction.Diapin was amidated by the method described in Bergstrom et al., J.Biol. Chem., 280: 23114-23121 (2005) and/or acetylated by the methoddescribed in John et al., Eur. J. Med. Res., 13: 73-78 (2008).

Effect of Amidation on Diapin

An experiment was performed in adult male C57BL/6J mice purchased fromJackson Lab. In the control group (diamond line in FIG. 15, n=10),glucose was orally administered at dose of 2 mg/g bw. In the Diapingroup (triangle line in FIG. 15, n=9), glucose and Diapin were orallyadministered at dose of 2 mg/g bw and 1 mg/g bw, respectively. In theamidated Diapin group (square line in FIG. 15, n=9), glucose andamidated Diapin were orally administered at dose of 2 mg/g bw and 1 mg/gbw, respectively. Blood glucose was measured at 0, 0.5, 1, 1.5 and 2hours after gavaging glucose, Diapin and amidated Diapin.

Blood glucose levels at 0, 0.5, 1, 1.5 and 2 hours in the Diapin groupand amidated Diapin group were significantly lower than those in thecontrol.

Effect of Acetylation of Diapin

An experiment was performed in adult male C57BL/6J mice purchased fromJackson Lab. In the Diapin group (diamond line in FIG. 16, n=10),glucose and Diapin were orally administered at dose of 2 g/kg bw and 1g/kg bw, respectively. In the acetylated Diapin group (square line inFIG. 16, n=10), glucose and acetylated Diapin were orally administeredat dose of 2 g/kg bw and 1 g/kg bw, respectively. Blood glucose wasmeasured at 0, 0.5, 1, 1.5 and 2 hours after gavaging glucose, Diapin,and acetylated Diapin.

Blood glucose levels at 0, 0.5, 1, 1.5 and 2 hours in the Diapin groupwere not significantly different from those in the acetylated Diapingroup.

Effect of Dual Modification on Diapin

An experiment was performed in adult male C57BL/6J mice purchased fromJackson Lab. In the Diapin group (triangle line in FIG. 17, n=9),glucose and Diapin were orally administered at dose of 2 g/kg bw and 1g/kg bw, respectively. In the amidated/acetylated Diapin group (squareline, n=9), glucose and amidated/acetylated Diapin were orallyadministered at dose of 2 g/kg bw and 1 g/kg bw, respectively. Bloodglucose was measured at 0, 0.5, 1, 1.5 and 2 hours after gavagingglucose, Diapin and amidated/acetylated Diapin.

Blood glucose levels at 0, 0.5, 1, 1.5 and 2 hours in the Diapin groupwere not significantly different from those in the amidated/acetylatedDiapin group.

Example 9

Diapin reduces blood glucose levels when orally administered prior toglucose administration.

Effect of Diapin Given at 30 Min Prior to Oral Glucose Administration

An experiment was performed in adult male C57BL/6J mice purchased fromJackson Lab. Fasted mice were given water (diamond line in FIG. 18,n=10) or Diapin (1 mg/g bw, square line in FIG. 18, n=10), then oralgavage glucose 2 g/kg bw after 30 min. Blood glucose was measured at 0,30, 60 and 120 min after giving glucose.

Blood glucose levels at 30, 60 and 120 min in the Diapin group weresignificantly lower than those in control group.

Effect of Diapin Given at 1 Hour Prior to Oral Glucose Administration

An experiment was performed in adult male C57BL/6J mice purchased fromJackson Lab. Fasted mice were given water (diamond line in FIG. 19,n=10) or Diapin (1 mg/g bw, square line in FIG. 19, n=10), then oralgavage glucose 2 g/kg bw after 1 hour. Blood glucose was measured at 0,30, 60 and 120 min after giving glucose.

Blood glucose levels at 30, 60 min in the Diapin group weresignificantly lower than those in control group.

Example 10

Dipeptide GG does not significantly reduce blood glucose levels inC57BL/6J mice after oral glucose administration

Effect of Diapin and Dipeptides GG and GL on Blood Glucose Level afterOral Glucose Administration

An experiment was performed in adult male C57BL/6J mice purchased fromJackson Lab. Fasted mice were given glucose 200 mg/kg bw (diamond linein FIG. 20, n=10) or glucose 200 mg/kg bw plus Diapin (square line inFIG. 20, 1 mg/g bw, n=10), GG (circle line in FIG. 20, 0.67 mg/g bw,n=10) or GL (triangle line in FIG. 20, 0.67 mg/g bw, n=10). Bloodglucose was measured at 30, 60, 90 and 120 min after giving glucose.Diapin served as a positive control in the experiment.

Diapin significantly reduced blood glucose levels at 30, 60, 90 and 120min. Peptide GL reduced the blood glucose level at 30 min while peptideGG did not significantly reduce blood glucose levels in comparison toDiapin.

Effect of Diapin and Dipeptide LG on Blood Glucose Level after OralGlucose Administration

An experiment was performed in adult male C57BL/6J mice purchased fromJackson Lab. Fasted mice were given glucose 200 mg/kg bw (triangle linein FIG. 21, n=10), glucose 200 mg/kg bw plus Diapin (square line in FIG.21, 1 mg/g bw, n=10), or LG (triangle line in FIG. 21, 0.67 mg/g bw,n=10). Blood glucose was measured at 30, 60, 90 and 120 min after givingglucose. Diapin served as a positive control in the experiments.

The dipeptide LG showed a more transient affect than did Diapin inreducing blood glucose levels. Diapin reduced blood glucose levels atall measured time points, whereas the dipeptide LG reduced blood glucoselevels only at 30 and 60 min.

Example 11

Diapin lowers blood glucose level in ob/ob mice [Liu et al., Diabetes,52(6):1409-16 (2003)] after oral glucose administration.

An experiment was performed in adult male B6.V-Lepob/J mice purchasedfrom Jackson Lab. The fasted mice were given glucose 2 mg/g (n=10) orglucose 2 mg/g bw plus Diapin (1 mg/g bw, n=10) by gavaging. Bloodglucose levels were measured at 30, 60, 90 and 120 min after givingglucose and Diapin.

As shown in FIG. 22, Diapin inhibits the increase of blood glucose afteroral load of glucose in ob/ob mice.

Example 12

Diapin also lowers blood glucose level in db/db mice [Chen et al., Cell,(3):491-495 (1996) and Hummel et al., Science, 153 (740):1127-1128(1966)] after oral glucose administration.

The experiment was performed in adult male BKS.Cg-m+/+Leprdb/J micepurchased from Jackson Lab. The fasted mice were given glucose 2 mg/g bw(n=10) or glucose 2 mg/g bw plus Diapin (1 mg/g bw, n=10) by gavaging.Blood glucose levels were measured at 30, 60, 90 and 120 min aftergiving glucose and Diapin.

As shown in FIG. 23, Diapin inhibits the increase of blood glucose afteroral load of glucose in db/db mice.

Example 13

Diapin lowers blood glucose level in high fat diet-induced diabetic mice[Tomas et al., Diabetes Obes. Metab., 13(1):26-33 (2011) and Dezaki etal., Diabetes, 55 (12):3486-93 (2006)] after oral glucoseadministration.

Wild type male C57BL/6J mice purchased from Jackson Lab were fed withhigh fat diet [rodent diet with 60% of calories from fat (Research DietsInc. Cat#: D12492)] for eight weeks to induce obesity with insulinresistance mouse model. Then, the fasted mice were given glucose 2 mg/gbw (n=10) or glucose 2 mg/g bw plus Diapin (1 mg/g bw, n=10) bygavaging. Blood glucose levels were measured at 30, 60, 90 and 120 minafter giving glucose and Diapin.

As shown in FIG. 24, Diapin inhibits the increase of blood glucose afteroral load of glucose in high fat diet-induced diabetic mice.

Example 14

Glycine-Glycine-D-Leucine (D-Diapin) has an extended effect on loweringblood glucose level in C57BL/6J mice after oral glucose administration.

An experiment was performed in adult male C57BL/6J mice purchased fromJackson Lab. The fasted mice were given glucose 2 mg/g bw (n=10) orglucose 2 mg/g bw plus D-Diapin (1 mg/g bw, n=10) or Diapin (1 mg/g bw,n=10) by gavaging. Blood glucose levels were measured at 30, 60, 90,120, and 180 min after giving glucose.

As shown in FIG. 25, D-Diapin is more effective than Diapin in loweringblood glucose levels after oral load of glucose in C57BL/6J mice.

Example 15

Diapin lowers blood glucose level in C57BL/6J mice after oral glucoseadministration and ip Diapin administration.

The experiment was performed in adult male C57BL/6J mice purchased fromJackson Lab. The fasted mice were given glucose 2 mg/g bw (n=14) orglucose 2 mg/g bw plus Diapin (1 mg/g bw, ip, n=14) or Diapin (1 mg/gbw, n=14) by gavaging. Blood glucose was measured at 30, 60, 90 and 120min after giving glucose and Diapin. Results are shown in FIG. 26.

Example 16

Modified dipeptides had different effects on blood glucose level inC57BL/6J mice after oral glucose administration. The modified dipeptidestested were an amidated GL dipeptide and the D-isomer of dipeptide LG.

An experiment was performed in adult male C57BL/6J mice purchased fromJackson Lab. The fasted mice were given glucose 2 mg/g bw (n=12) orglucose 2 mg/g bw plus Diapin (1 mg/g bw, ip, n=12) or dipeptide (0.67mg/g bw, n=12) by gavaging. Blood glucose was measured at 30, 60, 90 and120 min after giving glucose and peptide. Results are shown in FIG. 27.

While the present invention has been described in terms of specificembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Accordingly, only such limitations asappear in the claims should be placed on the invention.

All documents cited in this application are hereby incorporated byreference in their entirety for their disclosure described.

We claim:
 1. A method for treating a condition comprising administeringto a patient an effective amount of a composition, wherein thecomposition comprises: at least one peptide consisting of the amino acidsequence GGL, GLG, LGL, LLG, LGG, GLL, GGdL, GdLG, GdLL, GLdL, GdLdL,dLLG, LdLG, dLdLG, dLGG, dLGL, LGdL, or dLGdL, or a pharmaceuticallyacceptable salt of the peptide; at least one peptide consisting of theamino acid sequence GGL, GLG, LGL, LLG, LGG, GLL, GGdL, GdLG, GdLL,GLdL, GdLdL, dLLG, LdLG, dLdLG, dLGG, dLGL, LGdL, or dLGdL, or apharmaceutically acceptable salt of the peptide, wherein the peptide isacetylated at the N-terminus; at least one peptide consisting of theamino acid sequence GGL, GLG, LGL, LLG, LGG, GLL, GGdL, GdLG, GdLL,GLdL, GdLdL, dLLG, LdLG, dLdLG, dLGG, dLGL, LGdL, or dLGdL, or apharmaceutically acceptable salt of the peptide, wherein the peptide isamidated at the C-terminus; or at least one peptide consisting of theamino acid sequence GGL, GLG, LGL, LLG, LGG, GLL, GGdL, GdLG, GdLL,GLdL, GdLdL, dLLG, LdLG, dLdLG, dLGG, dLGL, LGdL, or dLGdL, or apharmaceutically acceptable salt of the peptide, wherein the peptide isacetylated at the N-terminus and amidated at the C-terminus; and whereinthe condition is prediabetes, diabetes, obesity, high blood pressure,metabolic syndrome, poor glycemic control, or reduced insulin secretion.2. The method according to claim 1, wherein the at least one peptideconsists of the amino acid sequence GGL, GLG, LGL, LLG, LGG, GLL, or anymixture thereof.
 3. The method according to claim 1, wherein the atleast one peptide consists of the amino acid sequence GGdL, GdLG, GdLL,GLdL, GdLdL, dLLG, LdLG, dLdLG, dLGG, dLGL, LGdL, dLGdL, or any mixturethereof.
 4. The method of claim 1, wherein the composition isadministered by an oral, intraperitoneal, ocular, intradermal,intranasal, subcutaneous, intramuscular or intravenous route.
 5. Themethod of claim 4, wherein the composition is administered by an oralroute.
 6. The method according to claim 2, wherein the condition isprediabetes or diabetes.
 7. The method according to claim 3, wherein thecondition is prediabetes or diabetes.
 8. The method according to claim2, wherein the condition is obesity, high blood pressure or metabolicsyndrome.
 9. The method according to claim 3, wherein the condition isobesity, high blood pressure or metabolic syndrome.
 10. The methodaccording to claim 2, wherein the at least one peptide consists of theamino acid sequence GGL.
 11. The method according to claim 3, whereinthe at least one peptide consists of the amino acid sequence GGdL.
 12. Amethod of preventing, reducing, or ameliorating a diabetes-associatedcomplication in a diabetic patient comprising administering to thepatient an effective amount of a composition, wherein the compositioncomprises: at least one peptide consisting of the amino acid sequenceGGL, GLG, LGL, LLG, LGG or GLL, GGdL, GdLG, GdLL, GLdL, GdLdL, dLLG,LdLG, dLdLG, dLGG, dLGL, LGdL or dLGdL, or a pharmaceutically acceptablesalt of the peptide; at least one peptide consisting of the amino acidsequence GGL, GLG, LGL, LLG, LGG, GLL, GGdL, GdLG, GdLL, GLdL, GdLdL,dLLG, LdLG, dLdLG, dLGG, dLGL, LGdL, or dLGdL, or a pharmaceuticallyacceptable salt of the peptide, wherein the peptide is acetylated at theN-terminus; at least one peptide consisting of the amino acid sequenceGGL, GLG, LGL, LLG, LGG, GLL, GGdL, GdLG, GdLL, GLdL, GdLdL, dLLG, LdLG,dLdLG, dLGG, dLGL, LGdL, or dLGdL, or a pharmaceutically acceptable saltof the peptide, wherein the peptide is amidated at the C-terminus; or atleast one peptide consisting of the amino acid sequence GGL, GLG, LGL,LLG, LGG, GLL, GGdL, GdLG, GdLL, GLdL, GdLdL, dLLG, LdLG, dLdLG, dLGG,dLGL, LGdL, or dLGdL, or a pharmaceutically acceptable salt of thepeptide, wherein the peptide is acetylated at the N-terminus andamidated at the C-terminus.
 13. The method according to claim 12,wherein the at least one peptide consists of the amino acids GGL, GLG,LGL, LLG, LGG or GLL, or any mixture thereof.
 14. The method accordingto claim 12, wherein the at least one peptide consists of the aminoacids GGdL, GdLG, GdLL, GLdL, GdLdL, dLLG, LdLG, dLdLG, dLGG, dLGL, LGdLor dLGdL, or any mixture thereof.
 15. The method of claim 12, whereinthe composition is administered by an oral, intraperitoneal, ocular,intradermal, intranasal, subcutaneous, intramuscular or intravenousroute.
 16. The method of claim 15, wherein the composition isadministered by an oral route.
 17. The method according to claim 13,wherein the diabetes-associated complication is a cardiovasculardisease, chronic kidney disease, kidney failure, bladder problems,erectile dysfunction, gastroporesis, an eye disease, a diabeticneuropathy, foot or skin ulcers, or lower extremity amputation.
 18. Themethod according to claim 14, wherein the diabetes-associatedcomplication is a cardiovascular disease, chronic kidney disease, kidneyfailure, bladder problems, erectile dysfunction, gastroporesis, an eyedisease, a diabetic neuropathy, foot or skin ulcers, or lower extremityamputation.
 19. The method according to claim 13, wherein the at leastone peptide consists of the amino acid sequence GGL.
 20. The methodaccording to claim 14, wherein the at least one peptide consists of theamino acid sequence GGdL.